In the production of ultra-thin biological specimen sections, especially for examination under a microscope an electron microscope, freezing of the specimen from which these sections are produced is frequently necessary. This is especially true with biological specimens having a high water content. It is of prime importance to be able to further maintain the frozen sections at very low temperatures, without any significant increase in temperature even for a short time. Also, care must be taken to prevent water condensing (or subliming) on the frozen section, since this would affect water-soluble components in the section and surface structure. Maintenance of the dry frozen condition must be uninterrupted right up to the final treatment or observation and frequently it must be continued during such a treatment or observation, for example, to prevent vaporization of substances contained in the specimen to be examined. Heating, which in the course of observations with an electron microscope can be caused by the electron beam and, at temperatures in the range of -70.degree. to -80.degree.C. would cause in recrystallization, making the specimen useless. In this connection, it should be mentioned that observation by an electron microscope requires the specimen to be located in an evacuated chamber enabling partial sublimation to occur at temperatures in the order of -90.degree.C. These difficulties are known, but the known methods and devices only partially overcome these problems and are also restricted to a few special uses.
Thus, ultramicrotomes with a freezing attachment are known and used to produce frozen sections. It is also known to use a carrier (which in itself is not frozen) for the transporting of a specimen to an electron microscope. Such carriers are pre-cooled to a low temperature but, in spite of the short time required for conveyance, the specimen is not adequately protected. Another uses a liquid-nitrogen filled, container as a carrier and the specimen floats on the liquid nitrogen as in an ultramicrotome.
It is also known that the stage of an electron microscope may be precooled and to use a specimen holder which forms part of the object stage as a transfer device. A device is known for transferring a frozen specimen located in a container of liquid nitrogen, in a dry nitrogen atmosphere, to the lock chamber of an electron microscope having a precooled stage. This device results in a brief by significant period of an elevated temperature having a deleterious effect on the specimen. Even this device does not solve the problems of removing and preserving a biological specimen section produced in the freezing chamber of an ultramicrotome in a dry, frozen state or the task of maintaining the specimen at a predetermined cryogenic temperature during further manipulations. Such devices do not prevent exposure for a short time to the ambient atmosphere during removal from the ultramicrotome.
It is an object of the present invention to provide a device capable of maintaining a biological section in a stable cryogenic environment during transfer from a sectioning instrument such as an ultramicrotome, or to a treatment or observation device.
It is another object of the present invention to provide an uncomplex, low cost device for transferring a section removed from the freezing chamber of an ultramicrotome inserting the device into the freezing chamber.
A further object of the present invention is a device which is self-cooled and temperature-regulated.
A still further object of the present invention is a vacuum chamber enabling the chamber and transfer device to form a separable unit, so that the transfer device therefore, with the section, can be conveyed without difficulty from one treatment or observation instrument to another, and coupled thereto. This is especially significant, if an intermediate treatment of the specimen section is to be conducted, as for instance etching, during which it must be possible to vary the temperature of the section.
It has been found that it is unnecessary to protect the specimen from water condensation by transfer in vacuum which has been frequently done. Rather it is sufficient to convey the section in a small convered recess in which is inserted inside of the freezing chamber of an ultramicrotome, having an inert, dry, cold gas atmosphere to receive the specimen.